Discovery and Structural Optimization of Spirodioxynaphthalene-Based Mdr1 Efflux Pump Inhibitor to Combat Azole Resistance in Candida albicans

Overexpression of efflux pump genes drives azole resistance in Candida albicans, highlighting the need for efflux pump inhibitors. We identified pleopcin A (PA), a novel spirodioxynaphthalene from Pleopunctum brunnescens, which reverses Mdr1-mediated azole resistance. To enhance potency, we achieved its first total synthesis and designed 82 derivatives via target–ligand analysis. Among them, PA36–2 emerged as the most potent lead compound, showing 64-fold greater activity than PA in combination with fluconazole against MDR1-overexpressing C. albicans strains. Mechanistic studies confirmed PA36–2 as a specific Mdr1 inhibitor. In addition, PA36–2 combined with fluconazole exhibited potent in vivo activity against Galleria mellonella and mice infected with MDR1-overexpressing C. albicans strain. These findings support further pharmaceutical investigation of the potential value of PA36–2 as an Mdr1 inhibitor for overcoming azole resistance in C. albicans infections.